The present invention relates to novel serine/threonine kinase receptor proteins, including a novel family of receptor proteins to bone morphogenetic proteins (BMPs). More particularly, the present invention relates to receptor proteins which are able to bind to BMPs, including BMP-2 and BMP-4. The present invention further relates to methods of isolating novel BMP receptor proteins using newly identified DNA fragments as probes for isolating such proteins.
Bone morphogenetic proteins (BMPs) are a family of proteins which have been identified as having the ability to induce the formation of bone and cartilage in tissue extracts. BMPs are a subfamily within the TGF-xcex2 superfamily. BMPs have multiple therapeutic uses, including a wide variety of settings where bone has been lost through physicological or traumatic processes.
The TGF-xcex2 superfamily of proteins have been shown to bind to serine/threonine kinase receptors. Massague, Cell, 69:1067-1070 (1992); Attisano et al., Cell 68:97-108 (1992); Lin et al., Cell, 68:775-785 (1992); Wang et al., Cell 67:797-805 (1991). Similarly, activin receptors have been isolated and characterized as a predicted transmembrane serine kinase. Mathews et al., Cell 65:973-982 (1991); Nakamura et al., J. Biol. Chem. 267:18924-18928 (1992): Ebner et al., Science, 260:1344-1348 (1993) describe the existence of Type I and Type II TGF-xcex2 receptors, and the effects of the Type I receptor on binding of TGF-xcex2 to the Type II receptor.
Type I receptor proteins have been reported not to bind to their ligand molecules independently, but, acting in concert with Type II receptor proteins, are observed to contribute to increased binding to the ligand. See Matsuzaki et al., J. Biol. Chem., 268:12719-12723 (1993); Ebner et al., Science, 260:1344-1348 (1993).
Paralkar et al., PNAS USA 88:3397-3401 (1991) describes the presence of high affinity binding sites for BMP-4 on MC3T3E1 and NIH3T3 cells. No competition by TGF-xcex2 was found for the BMP-4 binding proteins, nor was competition by BMP-4 for TGF-xcex2 receptors observed in Attisano et al., Cell 68:97-108 (1992).
In one embodiment, the present invention comprises a purified and isolated DNA molecule which encodes a BMP receptor protein, said DNA molecule preferably comprising the clones CFK1-43a and CFK1-23a, or a DNA sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:3.
The present invention further comprises purified and isolated DNA molecules which encode BMP receptor proteins, said BMP receptor proteins preferably comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:4. In another embodiment, the present invention comprises a BMP receptor protein CFK1-43a and CFK1-23a, comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:4.
The present invention further comprises DNA molecules comprising a DNA sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:7 and SEQ ID NO:9, and DNA molecules which encode serine/threonine kinase receptor proteins comprising an amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:8 and SEQ ID NO:10. These DNA molecules and proteins are related to the BMP family of receptors. Among other uses, these DNA molecules are presently useful as probes for isolating and purifying additional novel BMP receptors.
The present invention also comprises novel DNA sequences which encode receptor proteins, which novel DNA sequences are identified by a method using DNA sequence encoding all or a fragment of the receptor proteins of the present invention. In preferred embodiments, the novel DNA sequences are identified using DNA sequence from the serine/threonine kinase domain of a receptor, which is highly conserved among the family of BMP receptors. Alternatively, DNA sequence encoding the ligand binding domain could be used to identify additional novel BMP receptor encoding sequences.
The present invention further comprises DNA molecules encoding soluble, truncated receptor proteins, and the soluble proteins themselves. The truncated receptor proteins preferably comprise the ligand binding domain, but not the serine/threonine kinase and transmembrane domains, of the receptor protein. The truncated receptor proteins are soluble, and will be secreted into supernatant by mammalian cells. Thus, when expressed in mammalian cells using a DNA molecule encoding a truncated receptor protein, the truncated receptor protein will be secreted rather than expressed on the surface of the host cell. The truncated receptor protein thereby expressed still binds specifically to BMPs, and can be used to block receptors from mediating the cellular processes in which they normally participate in as signalling mechanisms by competition for the same ligand. The truncated receptor protein could compete with receptor proteins normally expressed on the surface of responsive cells for functional ligand and inhibit the formation of a functional receptor-ligand complex, thereby blocking the normal signalling mechanism of the complex and the cellular processes normally affected by functional receptor-ligand interactions.
In one aspect, the invention provides a method for producing cells expressing more than one receptor protein comprising culturing a selected host cell containing a polynucleotide sequence encoding a first selected receptor protein, truncated receptor protein, or active fragment thereof and a polynucleotide sequence encoding a second selected receptor protein, truncated receptor protein, or active fragment thereof. The resulting cells, which will express multiple co-expressed, biologically active receptors, may be isolated and used in a therapeutic composition.
Another aspect of the current invention comprises ligands for the BMP receptors and truncated BMP receptor protein, said ligands being characterized by the ability to bind to the receptors. Such ligands may stimulate growth of bone and/or cartilage, or may be involved in influencing other developmental processes. Said ligands may be monoclonal antibodies, small peptide BMP analogues, or small organic molecule BMP analogues as further characterized herein. In a preferred embodiment, said ligands comprise antibodies against the truncated, soluble receptor protein and the receptor proteins of the invention. These antibodies can be employed in a variety of diagnostic and therapeutic applications. Such antibodies can be used to identify cell types which naturally express receptors of the invention and may therefore have the capacity to elicit a biological response upon exposure to the appropriate ligand. These antibodies can be further useful in the identification of additional receptor proteins capable of binding to other individual BMPs and/or BMP heterodimers. Additionally such antibodies are useful in blocking the formation of functional receptor-ligand complexes and thus inhibit the cellular responses that would normally be mediated by these complexes. Alternatively, such antibodies may mimic the effect of BMP by interacting with the receptor in a way that would stimulate the cellular responses that would normally be mediated by a functional receptor-ligand complex.
In yet another embodiment, the invention comprises pharmaceutical compositions comprising a compound first identified for such use as a ligand for the truncated BMP receptor and therapeutic methods for the treatment of bone and/or cartilage disorders comprising administering a ligand for the truncated BMP receptor.
Other aspects and advantages of the present invention will be apparent upon consideration of the following detailed description and preferred embodiments thereof.